We have continued to characterize 2 flat cellular revertants, C-11 and F-2, which were originally derived from the DT line of Ki-MuSV-transformed NIH/3T3 cells. A third revertant, Clone 22 was isolated during the last year. The revertant phenotype can be transferred by transfection of purified DNA from clone 22 to recipient Ki-MuSV-transformed cells. These primary transfectants, after repeated biological cloning, exhibit a stable nontransformed phenotye but are resistant to challenge with both v-Ki-ras and v-Ha-ras. A rudimentary "restriction map" for the putative revertant gene was derived by quantitating the effect of 8 different restriction endonucleases on the activity of revertant DNA as measured by transfection of the revertant phenotype into the DT cell line. We have initiated studies designed to reveal the mechanism responsible for the revertant phenotype. The resistance of the C-11 and F-2 revertants to the toxic effects of ouabain, together with our previous ion transport data, suggested that these cells were altered in some aspect of ion transport. Initial results indicated that the activity of the enzyme Na+,K+ ATPase, the specific target of ouabain, was the same in the NIH/3T3, DT and revertant cell lines, both in the presence and absence of ouabain. Preliminary studies using Rb86 led to the same conclusion. We are currently studying the other major ion transport pathways in the revertant cell lines. In a collaborative project with Dr. Cooper, we have analyzed extracts from normal, transfomed and revertant cell lines by 2-dimensional gel electrophoresis. The synthesis of only 2 proteins, with molecular weights of 38,000 and 41,000, consistently change with the ras-induced transformed phenotype. These proteins are present in normal NIH/3T3 cells, disappear in ras-transformed cells, and reappear in revertants. The proteins have both been identified as forms of the cytoskeleton-associated tropomyosin family. Additional studies indicate that the loss of tropomyosin is confined to cells transformed by retroviruses.